In this study the tonB2 gene was cloned from Actinobacillus pleuropneumoniae JL01 (serovar 1) and expressed as a glutathione-S-transferase (GST) fusion protein in Escherichia coli BL21(DE3). The GST fusion protein was recognized by antibodies in serum positive for A. pleuropneumoniae by Western blot analysis. Purified soluble GST-TonB2 was assessed for its ability to protect BALB/c mice against A. pleuropneumoniae infection. Mice were vaccinated with GST-TonB2 subcutaneously and challenged intraperitoneally with either approximately 4.0 × 10(5) colony-forming units (CFU) or approximately 1.0 × 10(6) CFU of A. pleuropneumoniae 4074. They were examined daily for 7 d after challenge. The survival rate of the TonB2-vaccinated mice was significant higher than that of the mice given recombinant GST or adjuvant alone. These results demonstrate that A. pleuropneumoniae TonB2 is immunogenic in mice and should be further assessed as a potential candidate for a vaccine against A. pleuropneumoniae infection. In addition, an indirect enzyme-linked immunosorbent assay (ELISA) based on the GST-TonB2 recombinant protein was developed. Compared with the ApxIVA ELISA, the TonB2 ELISA provided earlier detection of antibodies in pigs at various times after vaccination with A. pleuropneumoniae live attenuated vaccine. When compared with an indirect hemagglutination test, the sensitivity and specificity of the TonB2 ELISA were 95% and 88%, respectively. The TonB2 ELISA provides an alternative method for rapid serologic diagnosis of A. pleuropneumoniae infection through antibody screening, which would be especially useful when the infection status or serovar is unknown.

Mitochondrial transcription factor A (Tfam) has been implicated in the pathogenesis of retinal dysplasia in miniature schnauzer dogs and it has been proposed that affected dogs have altered mitochondrial numbers, size, and morphology. To test these hypotheses the Tfam gene of affected and normal miniature schnauzer dogs with retinal dysplasia was sequenced and lymphocyte mitochondria were quantified, measured, and the morphology was compared in normal and affected dogs using transmission electron microscopy. For Tfam sequencing, retina, retinal pigment epithelium (RPE), and whole blood samples were collected. Total RNA was isolated from the retina and RPE and reverse transcribed to make cDNA. Genomic DNA was extracted from white blood cell pellets obtained from the whole blood samples. The Tfam coding sequence, 5' promoter region, intron1 and the 3' non-coding sequence of normal and affected dogs were amplified using polymerase chain reaction (PCR), cloned and sequenced. For electron microscopy, lymphocytes from affected and normal dogs were photographed and the mitochondria within each cross-section were identified, quantified, and the mitochondrial area (μm2) per lymphocyte cross-section was calculated. Lastly, using a masked technique, mitochondrial morphology was compared between the 2 groups. Sequencing of the miniature schnauzer Tfam gene revealed no functional sequence variation between affected and normal dogs. Lymphocyte and mitochondrial area, mitochondrial quantification, and morphology assessment also revealed no significant difference between the 2 groups. Further investigation into other candidate genes or factors causing retinal dysplasia in the miniature schnauzer is warranted.

Objective—To evaluate serum haptoglobin concentration at feedlot arrival and subsequent performance and morbidity and mortality rates of calves that developed bovine respiratory disease. Animals—360 heifer calves and 416 steer and bull calves. Procedures—Serum samples were obtained from cattle at the time of arrival to a feedlot (day −1) and analyzed for haptoglobin concentration. In experiment 1, calves were classified into groups with a low (< 1.0 μg/mL), medium (1.0 to 3.0 μg/mL), or high (> 3.0 μg/mL) serum haptoglobin concentration and allotted into pens on the basis of group. In experiment 2, calves were classified as having or not having detectable serum haptoglobin concentrations. Results—In experiment 1, average daily gain from days 1 to 7 decreased as haptoglobin concentration increased. Dry-matter intake (DMI) from days 1 to 21 decreased with increasing haptoglobin concentration, and DMI typically decreased from days 1 to 63. Total bovine respiratory disease morbidity rate typically increased with increasing haptoglobin concentration. At harvest, no differences in carcass characteristics were observed on the basis of haptoglobin concentration. In experiment 2, cattle with measureable serum haptoglobin concentrations at arrival weighed less throughout the experiment, gained less from days 1 to 7, and had lower DMI from days 1 to 42. Overall morbidity rate was not different between groups, but cattle with detectable serum haptoglobin concentrations had higher odds of being treated 3 times. Conclusions and Clinical Relevance—Serum haptoglobin concentration in cattle at the time of feedlot arrival was not associated with overall performance but may have limited merit for making decisions regarding targeted prophylactic treatment.

Objective—To evaluate enterotoxin production, enterotoxin gene distribution, and genetic diversity of Staphylococcus aureus in milk obtained from cows with subclinical mastitis. Sample—Milk samples obtained from 350 cows (1,354 mammary glands) on 11 Wisconsin dairy farms. Procedures—Of 252 S aureus isolates obtained from 146 cows, 83 isolates (from 66 cows with subclinical mastitis) were compared genotypically by use of pulsed-field gel electrophoresis and via PCR identification of toxic shock syndrome toxin 1 (TSST-1) and classical S aureus enterotoxin genes (sea, seb, sec, sed, and see). Results—Among the 83 S aureus isolates, ≥ 1 enterotoxin genes were identified in 8 (9.6%). Enterotoxin gene distribution was as follows: TSST-1, 7 isolates (8.4%); sec, 5 isolates (6.0%); and sed, 2 isolates (2.4%). Enterotoxin genes sea, seb, and see were not identified. Twelve pulsotypes and 5 subtypes were identified among the 83 isolates; 5 of the 12 pulsotypes were represented by only 1 isolate. In cows of 1 herd, only a single S aureus pulsotype was detected; in cows on most other farms, a variety of pulsotypes were identified. One pulsotype was recovered from 4 farms (n = 23 cows) and another from 5 other farms (16). Isolates with an enterotoxin gene were represented by 6 pulsotypes. Conclusions and Clinical Relevance—S aureus classical enterotoxins and TSST-1 were rarely recovered from milk samples obtained from cows with subclinical mastitis in Wisconsin. Diverse pulsotypes of S aureus were detected within and among farms, indicating that different strains of S aureus cause subclinical mastitis in dairy cows.

Objective—To assess expression pattern and subcellular compartmentalization of 5-lipoxygenase in cutaneous, UV radiation–induced, and oral squamous cell carcinomas (SCCs) in cats and determine the effects of cyclooxygenase or 5-lipoxygenase inhibition on proliferation or apoptosis in a feline oral squamous cell carcinoma (SCCF1) cell line. Sample—60 archived paraffin-embedded samples of SCCs from 60 cats and SCCF1 cells. Procedures—Retrospective immunohistochemical analysis of the archived samples of SCCs (20 cutaneous, 20 UV radiation–induced, and 20 oral tumors) was performed. Cell culture proliferation assays involving SCCF1 cells were performed, and tepoxalin-induced apoptosis and signaling were examined via western blotting and annexin V staining. Results—Immunohistochemically, staining for 5-lipoxygenase was most frequently of greatest intensity in oral SCCs, whereas staining of cutaneous and UV radiation–induced lesions had less consistent 5-lipoxygenase expression. Exposure of SCCF1 cells to the 5-lipoxygenase inhibitor tepoxalin resulted in apoptosis; the effect appeared to be mediated via alteration of cell signaling rather than via suppression of lipid mediators that are typically produced as a result of 5-lipoxygenase activity. Conclusions and Clinical Relevance—In cats, expression of 5-lipoxygenase in SCCs appeared to differ depending on tumor location. The influence of tepoxalin-induced 5-lipoxygenase inhibition on a poxygenase–expressing cell line coupled with the notable expression of 5-lipoxygenase in oral SCCs suggested that 5-lipoxygenase inhibition may have therapeutic benefits in affected cats. Although the safety of tepoxalin in cats has yet to be investigated, 5-lipoxygenase inhibitors should be evaluated for use as a potential treatment for SCCs in that species.

Objective-To determine variations in cytologic counts of bronchoalveolar lavage (BAL) fluid attributable to month of collection, first and second aliquots, and left and right lung sites in horses with recurrent airway obstruction (RAO). Animals-5 horses with RAO and 5 healthy horses without respiratory tract disease. Procedures-Horses were housed in a stable for 5 months prior to and throughout the study. Bronchoalveolar lavage fluid was collected from the right and left lung of each horse 3 times at monthly intervals (February, March, and April). Each BAL fluid collection was performed by use of 2 incremental instillations of 250 mL of isotonic saline (0.9% NaCl) solution in the same bronchial site. Analysis of BAL fluid included volume of BAL fluid recovered, a CBC, and differential cytologic counts. Results-Volume of BAL fluid recovered and cytologic counts did not differ in horses with RAO across time or between right and left lungs, except for the number of mast cells. Horses with RAO had significantly lower volumes of BAL fluid recovered, significantly lower percentages of macrophages and lymphocytes, and significantly higher percentages of neutrophils than did healthy horses. Despite individual variation, all horses with RAO had > 25% neutrophils throughout the study period. Conclusions and Clinical Relevance-Despite variation among horses, BAL fluid cytologic counts were repeatable over short and long periods and samples can be used for longitudinal studies as a diagnostic tool of pulmonary inflammation in horses with RAO.

Objective—To compare echocardiographic indices of myocardial strain with invasive measurements of left ventricular (LV) systolic function in anesthetized healthy dogs. Animals—7 healthy dogs. Procedures—In each anesthetized dog, preload and inotropic conditions were manipulated sequentially to induce 6 hemodynamic states; in each state, longitudinal, radial, and global strains and strain rate (SR), derived via 2-D speckle-tracking echocardiography, were evaluated along with conventional echocardiographic indices of LV function and maximum rate of rise (first derivative) of LV systolic pressure (LV+dp/dtmax). Catheter-derived and echocardiographic data were acquired simultaneously. Partial and semipartial correlation coefficients were calculated to determine the correlation between LV+dp/dtmax and each echocardiographic variable. Global longitudinal strain was compared with conventional echocardiographic indices via partial correlation analysis. Results—All myocardial segments could be analyzed in all dogs. Significant semipartial correlations were identified between conventional echocardiographic strain indices and LV+dp/dtmax. Correlation coefficients for longitudinal deformation and global strain, segmental longitudinal strain, and segmental SR were −0.773, −0.562 to −0.786, and −0.777 to −0.875, respectively. Correlation coefficients for radial segments and strain or SR were 0.654 to 0.811 and 0.748 to 0.775, respectively. Correlation coefficients for traditional echocardiographic indices and LV+dp/dtmax (−0.586 to 0.821) and semipartial correlation coefficients for global strain and echocardiographic indices of LV systolic function (−0.656 [shortening fraction], −0.726 [shortening area], and −0.744 [ejection fraction]) were also significant. Conclusions and Clinical Relevance—Results indicated that LV systolic function can be predicted by myocardial strain and SR derived via 2-D speckle-tracking echocardiographic analysis in anesthetized healthy dogs.

Objective—To investigate the in vitro effect of the combination of lignan enterolactone (ENL) or lignan enterodiol (END) with melatonin on steroid hormone secretion and cellular aromatase content in human adrenal carcinoma cells. Sample—Human adrenocortical carcinoma cells. Procedures—Melatonin plus ENL or END was added to cell culture medium along with cAMP (100μM); control cells received cAMP alone. Medium and cell lysates were collected after 24 and 48 hours of cultivation. Samples of medium were analyzed for progesterone, 17-hydroxyprogesterone, androstenedione, aldosterone, estradiol, and cortisol concentration by use of radioimmunoassays. Cell lysates were used for western blot analysis of aromatase content. Results—The addition of ENL or END with melatonin to cAMP-stimulated cells (treated cells) resulted in significant decreases in estradiol, androstenedione, and cortisol concentrations at 24 and 48 hours, compared with concentrations in cells stimulated with cAMP alone (cAMP control cells). The addition of these compounds to cAMP-stimulated cells also resulted in higher progesterone and 17-hydroxyprogesterone concentrations than in cAMP control cells; aldosterone concentration was not affected by treatments. Compared with the content in cAMP control cells, aromatase content in treated cells was significantly lower. Conclusions and Clinical Relevance—The combination of lignan and melatonin affected steroid hormone secretion by acting directly on adrenal tumor cells. Results supported the concept that this combination may yield similar effects on steroid hormone secretion by the adrenal glands in dogs with typical and atypical hyperadrenocorticism.

Objective—To characterize the effects of pregnancy on insulin sensitivity (SI) and glucose dynamics in pasture-maintained mares fed supplemental feeds of differing energy composition. Animals—Pregnant (n = 22) and nonpregnant (10) healthy Thoroughbred mares. Procedures—Pregnant and nonpregnant mares underwent frequently sampled intravenous glucose tolerance tests at 2 times (period 1, 25 to 31 weeks of gestation; period 2, 47 weeks of gestation). Following period 1 measurements, mares were provided a high-starch (HS; 39% starch) or high-fat and -fiber (14% fat and 70% fiber) supplemental feed. From a subset of mares (n = 12), blood samples were collected hourly for 24 hours to assess glycemic and insulinemic response to feeding while pastured. The minimal model of glucose and insulin dynamics was used to estimate SI, glucose effectiveness, and acute insulin response to glucose from tolerance testing data. Results—Pregnant mares during period 1 had a lower SI and glucose effectiveness and higher acute insulin response to glucose than did nonpregnant mares. The SI value decreased in nonpregnant but not pregnant mares from periods 1 to 2. Pregnant mares fed HS feed had a greater glycemic and insulinemic response to feeding than did any other group. Conclusions and Clinical Relevance—Pregnant mares had slower glucose clearance and greater insulin secretion at 28 weeks of gestation than did nonpregnant mares. Glucose and insulin responses to meal feeding, particularly with HS feed, were greater in pregnant mares, indicating that pregnancy enhanced the postprandial glycemic and insulinemic effects of starch-rich feed supplements.

Objective—To induce ischemia and reperfusion injury in the large colon mucosa of horses in vivo and evaluate the recovery and effects of components of an organ transplant solution on mucosal recovery in vitro. Animals—6 healthy horses. Procedures—Horses were anesthetized, and ischemia was induced for 60 minutes in the pelvic flexure, which was followed by reperfusion for 240 minutes. Ischemic (n = 4 horses), reperfused (6), and adjacent control (6) colonic mucosae were isolated for in vitro testing and histologic examinations. Tissues were mounted in Ussing chambers with plain Krebs Ringer bicarbonate (KRB), KRB with N-acetylcysteine (NAC), or KRB with a modified organ transplant solution (MOTS). Transepithelial electrical resistance (TER) and mannitol flux were used to assess mucosal integrity. Data were analyzed by use of ANOVA and Kruskal-Wallis tests. Results—The TER in reperfused tissues was similar to the TER in control tissues and greater than the TER in ischemic tissues, which was consistent with morphological evidence of recovery in reperfused tissues. Mannitol flux was greater in ischemic tissues than in reperfused tissues. The TER and mannitol flux were not significantly affected by incubation of mucosa with NAC or MOTS. Conclusions and Clinical Relevance—Ischemia induced during the brief period allowed rapid mucosal repair and complete recovery of tissue barrier properties during reperfusion. Therefore, reperfusion injury was not observed for this method of ischemic damage in equine colonic mucosa.